Our long-term goal is to understand the relationship between metabolism and neuronal excitability and to investigate how this relationship can be altered in diseased states such as in epilepsy. Our immediate goal is to understand how compartmentation of the key metabolite ATP can modulate neuronal excitability. Energy metabolism and ATP-dependent processes are vital to all mammalian cells. A long-standing and still unresolved hypothesis is that ATP compartments exist within the cytoplasm. Evidence suggests that ATP compartmentation could be critical to the regulation of chemical and electrical signaling in many cell types, but this hypothesis is controversial. Resolution of this controversy would provide a significant advance in our basic understanding of intracellular signaling, and it has implications for our understanding of health problems such as diabetes, ischemic injuries, and epilepsy. In neurons specifically, compartmentation of ATP could be a critical factor affecting plasticity and membrane excitability. ATP compartmentation may occur because of the specialized geometry of neurons whose neurites extend far from the cell body. For example, high metabolic requirements and local ATP consumption in dendritic compartments may affect synaptic plasticity. In another scenario, restricted diffusion of ATP between the bulk cytoplasm and near the plasma membrane (the "submembrane" compartment) may impact excitability. The Na,K-ATPase is a major energy consumer in neurons, and pump activation following neuronal activity may deplete submembrane ATP. Neuronal ATP-sensitive potassium channels (KATP channels) are sensitive to submembrane ATP and ADP and could control excitability through a negative feedback loop. Although experiments using electrophysiology, biochemistry, and cell biology support an important role for ATP compartmentation, there is a lack of direct evidence. To directly investigate ATP compartmentation, better optical tools are needed for imaging intracellular ATP. Therefore, during this fellowship I will investigate ATP compartmentation in neurons with three specific aims: (1) I will develop methods for imaging the ATP-to-ADP ratio in neurons using an improved genetically-encoded, ratiometric fluorescent sensor that is targeted to subcellular locations. (2) I will investigate how ATP levels respond to neuronal activation and whether ATP is compartmented locally within dendrites or between the bulk cytoplasm and a submembrane space. (3) I will investigate how ATP levels respond to a change in fuel source and whether choice of fuel affects ATP compartmentation between the cell body, submembrane compartment, and dendrites. Using fluorescence microscopy to investigate these specific aims, I will be able to study how ATP compartmentation acts as a critical parameter in modulating neuronal excitability. PUBLIC HEALTH RELEVANCE: Metabolism can regulate the excitability of neurons, and this relationship has been exploited to treat diseases such as epilepsy through diet. ATP compartmentation in neurons could be critical to metabolic regulation, and investigation of compartmentation is necessary to fully understand normal versus diseased states and to design molecular therapies.